Alejandra M. G. Del Carpio

Enteroaggregative Escherichia coli (EAEC) is one of the main diarrhea causative agents. This E. coli pathotype is defined by an aggregative adherence pattern observed in HEp-2 cells. Molecular diagnosis is still a challenge due to EAEC genetic heterogeneity. Different publications described primers for aggR and aatA detection to characterize EAEC. Recently, afpR has been suggested as EAEC molecular marker of some strains, demonstrating the existence of two subpopulations within this pathotype: typical and atypical EAEC, respectively, comprising aatA +/ aggR + and aatA +/ afpR +. Studies performed by our group, however, detected conflicting results using different primer pairs for aggR . Therefore, this study mainly aimed to analyze the variation of aggR sequences and its implication in EAEC characterization, defining consensus primers. As a consequence, a triplex-PCR targeting aggR , aatA, and afpR is proposed as a one-step molecular diagnosis for both typical and atypical EAEC. Previously reported primer sequences used for aggR, aatA , and afpR detection, and their annealing sites were analyzed using sequence databases. The best primers for each target were selected, and a triplex-PCR was standardized and validated. Database alignment of aggR revealed numerous variant regions along its sequence, impairing the annealing of most reported primers, while significant variations in the annealing sites of aatA and afpR primers were not observed. Broad-range primers for aggR were determined and combined with aatA and afpR , resulting in a triplex-PCR. Our triplex-PCR targeting aggR, aatA , and afpR successfully detected only EAEC, covering all aggR variants, promoting a broad and specific detection of this pathotype. IMPORTANCE: Phenotypical and genotypical characterization of enteroaggregative Escherichia coli (EAEC) is either time-consuming or of variable sensitivity. Therefore, developing an accurate molecular diagnostic method for this E. coli pathotype is relevant for public health. This study analyzed the primers currently used to detect typical EAEC-related genes ( aatA and aggR ), and afpR as a marker for atypical EAEC. After an extensive literature review, it was observed that the aatA primers are suitable for its broad detection. On the other hand, none of the aggR primer pairs described were able to detect all variants of the aggR gene. To address this issue, universal primers for aggR were proposed to improve the detection of all aggR variants. Considering the demand for a fast and accurate molecular approach for EAEC diagnosis, a triplex-PCR assay was developed, validated, and optimized using the universal aggR primers in combination with primers for aatA and afpR , detecting both typical and atypical EAEC.

The functions of bacterial plasmid-encoded toxin–antitoxin (TA) systems are unambiguous in the sense of controlling cells that fail to inherit a plasmid copy. However, its role in chromosomal copies is contradictory, including stress-response-promoting fitness and antibiotic treatment survival. A hybrid pathogenic Escherichia coli strain may have the ability to colonize distinct host niches, facing contrasting stress environments. Herein, we determined the influence of multiple environmental stress factors on the bacterial growth dynamic and expression profile of previously described TA systems present in the chromosome of a hybrid atypical enteropathogenic and extraintestinal E. coli strain. Genomic analysis revealed 26 TA loci and the presence of five type II TA systems in the chromosome. Among the tested stress conditions, osmotic and acid stress significantly altered the growth dynamics of the hybrid strain, enhancing the necessary time to reach the stationary phase. Using qPCR analyses, 80% of the studied TA systems were differentially expressed in at least one of the tested conditions, either in the log or in the stationary phase. These data indicate that type II TA systems may contribute to the physiology of pathogenic hybrid strains, enabling their adaptation to different milieus.

Escherichia coli is a frequent pathogen isolated from bloodstream infections. This study aimed to characterize the genetic features of EC092, an E. coli strain isolated from bacteremia that harbors enteroaggregative E. coli (EAEC) genetic markers, indicating its hybrid pathogenic potential. Whole-genome sequencing showed that EC092 belongs to phylogroup B1, ST278, and serotype O165:H4. Genes encoding virulence factors such as fimbriae, toxins, iron-uptake systems, autotransporter proteins (Pet, Pic, Sat, and SepA), and secretion systems were detected, as well as EAEC virulence genes (aggR, aatA, aaiC, and aap). EC092 was found to be closely related to the other EAEC prototype strains and highly similar in terms of virulence to three EAEC strains isolated from diarrhea. The genomic neighborhood of pet, pic, sat, sepA, and the EAEC virulence genes of EC092 and its three genetically related fecal EAEC strains showed an identical genomic organization and nucleotide sequences. Also, EC092 produced and secreted Pet, Pic, Sat, and SepA in the culture supernatant and resisted the bactericidal activity of normal human serum. Our results demonstrate that the strain EC092, isolated from bacteremia, is a hybrid pathogenic extraintestinal E. coli (ExPEC)/EAEC with virulence features that could mediate both extraintestinal and intestinal infections.